As self-organizing noncovalent supramolecular assemblies, membranes have offered a model for molecular engineering. Phospholipids differing in chemical composition, saturation, and size have been utilized as building blocks in the design of lipid-based cylinders, cubes, and spheres which have found applications both in drug delivery and as templates for composite structures [Fuhrhop et al. in Membranes and Molecular Assemblies: Synkinetic Approach, (1994) Royal Society of Chemistry, Cambridge, UK]. Likewise, recent approaches directed at the design of advanced materials for tissue engineering have emphasized the adhesion of cells on synthetic surfaces functionalized with epitopes derived from extracellular matrix proteins [Martin et al. (1982) J. Biol. Chem. 257:286-288; Shek et al. (1983) Immunology 50:101-106]. Polymerizable lipid amphiphiles, comprising lipids modified to contain reactive groups in distinct locations, provide opportunities to prepare such novel materials useful in diagnostics, drug delivery, surface modifications, etc. [O'Brien, Trends in Polymers (1994) 2:183-188].
Biomolecular recognition in multicellular systems is achieved by the presence of both integral proteins and carbohydrates which act as either ligands or receptors for neighboring cells, matrix, or soluble factors [Yeagle (1992) "The Structure of Biological Membranes," CRC Press, Boca Raton, Fla., USA]. Optimization of these receptor-ligand interactions is predicated upon lateral and rotational diffusion of both lipids and other macromolecules within the membrane suprastructure. Together, these structural features dictate, at least in part, the interaction of a cell with its surrounding microenvironment-interactions which bioengineers have sought to mimic in the rational design of biologically functional materials.
There are several classes of membrane lipids. The most abundant membrane lipids are the polar phospholipids, containing phosphorus in the form of phosphoric acid groups. The major phospholipids found in membranes are the phosphoglycerides, which contain two fatty acid molecules esterified to the first and second hydroxyl groups of glycerol. The third hydroxyl group of glycerol forms an ester linkage with phosphoric acid. The most abundant phosphoglycerides are the closely related phosphatidylethanolamine, phosphatidylcholine, phosphatidylserine and phosphatidylinositol. Phosphoglycerides are amphipathic, having a polar, hydrophilic "head" (phosphoric) group and nonpolar, hydrophobic tails.
Methods for synthesizing glycophospholipid conjugates are known in the literature. In some methods, a saccharide is combined with a phosphatidyl nitrogenous base through reductive amination. For example, a saccharide, in the form of a reducing sugar, is converted into a corresponding alditol by periodate oxidation and conjugated to a phosphatidylethanolamine derivative to form an imine intermediate, which is then reduced in the presence of sodium cyanoborohydride to produce a stable amino linkage [Tang et al. (1985) Biochem. Biophys. Res. Comm., 132:474-480]. The problem associated with this method of synthesizing glycophospholipid conjugates is that the ring structure of the reducing terminus of the saccharide is destroyed. Destruction of a functional group in the saccharide structure lowers the reactivity of the saccharide molecule and leads to changes in the biological activity of any glycolipid conjugates produced therefrom. Another problem with this method of synthesis is the lack of economy and/or efficiency between reactant equivalences in the lipid-saccharide conjugation reaction.
Glycolipid conjugates have also been prepared biochemically using enzyme preparations. For example, a specific type of phospholipase D [Kokusho et al., U.S. Pat. No. 4,624,919, issued Nov. 25, 1986; Shuto et al. (1988) Chem. Pharm. Bull., 36:5020-5023] has been used to facilitate the transfer of the phosphatidyl moiety from phosphatidylcholine to a primary hydroxyl group of a saccharide. However, the problem associated with this biochemical method of preparing glycophospholipid conjugates is that this method allows the preparation of only glycophospholipid conjugates having a fixed, non-modifiable distance between the phospholipid moiety and the saccharide residue. Undue proximity between the phospholipid and the sugar residues exerts stearic limitations and possible hindrance with respect to utilities associated with incorporation of synthesized glycolipids into membrane structures and their biological functionings.
The synthesis of a phospholipid-galactose conjugate has been reported by Haensler et al. (1991) (Glycoconjugate J. 8:116-124). This method involves preparing 2'-carboxyethyl-1-thiogalactoside in four steps from commercially available peracetylated galactose. The carboxylic acid on the galactoside is further refunctionalized with 1,3-diamino-2-propanol via amidation. The derivatized galactose is then coupled to the N-hydroxysuccinimide ester of N-succinyl phosphatidylethanolamine. The linker between the phospholipid and the galactose in the final product contains a minimum of sixteen hydrocarbon, carbonyl and amide bonds. This procedure is not suitable if conjugates having a shorter linker are desired.
In other art, dicarboxymethylated glycolipids were prepared by methods known in the art for use as cell adhesion inhibitors [Martel et al. EP 07197871]. In yet other art, ether lipid-nucleoside conjugates have been prepared and used to combat HIV-1 infections (Piantadosi et al., U.S. Pat. No. 5,512,671, Apr. 30, 1996).
Phospholipid-saccharide conjugates were also produced by the reaction of a phospholipid derivative with an activated saccharide to form a phospholipid-saccharide molecule joined by a diether linkage (Staveski et al., U.S. Pat. No. 5,354,853, issued Oct. 11, 1994). The diether linkage bridging the phospholipid and saccharide comprised a straight chain or branched alkyl group having from 1 to about 20 carbon atoms. However, one of the problems associated with this method is the extremely low yield of phospholipid-saccharide conjugate obtained by this process. For example, the yields obtained for the preparation of phospholipid-monosaccharide conjugates were in the range of 0.7 to 6% of the starting amount of protected monosaccharide. Additionally, this method required a long time (approximately 4 days) for the preparation of a phospholipid-saccharide conjugate.
Methods are also available in the art for the synthesis of peptide-phospholipid conjugates, e.g., with enzymatic transphosphatidylation [Wang et al. (1993) J. Am. Chem. Soc. 115:10487-10491]. However, these methods present problems with lack of economy, efficiency and low yields, similar to those associated with the prior art methods for glycophospholipid conjugates.
Thus, there is a need in the art for glycophospholipid and protein-phospholipid conjugates wherein the saccharide ring structure remains functionally intact and wherein the linkage joining the phospholipid and saccharide or peptide moieties constitutes a flexible spacer arm having a modifiable length and composition. There also exists a need for a method of synthesis of such glycophospholipid or peptide-phospholipid conjugates, wherein the method is highly efficient and economical and provides high yields of conjugate products and wherein the method is carried out easily under routine laboratory conditions.
These needs in the art are met by the present invention as disclosed herein.